The phytoestrogen genistein (NSC 36586) is being considered as a potential agent for the prevention and treatment of cancer. Although its pharmacological properties have been copiously studied for many decades, a search of the literature revealed that the disposition and bioavailability of genistein has not been characterized in any species. Furthermore, detailed consideration of the methods previously used to assay genistein in biological fluids revealed that they would be unsuitable for pharmacokinetic drug level monitoring in small animals. Although allowing genistein to be measured at concentrations near 1 to 2 ng/mL, extremely large sample volumes (4-100 mL) were required to achieve this degree of sensitivity. The work presently described was undertaken to develop a technically convenient assay specifically for unchanged genistein in plasma and urine employing a sample volume to facilitate replicate analysis of specimens acquired from individual mice. This was achieved by a procedure involving isocratic high performance liquid chromatography with ultraviolet detection following isolation of genistein from the biological matrix by quantitative extraction into an immiscible organic solvent. The assay has been thoroughly validated and proven highly reliable. The maximum dose of genistein given to mice by rapid intravenous (iv) injection that was completely nontoxic was near 50 mg/kg, whereas oral (po) doses as large as 500 mg/kg were apparently nontoxic. The compound was rapidly eliminated from mice and dogs following iv injection, with total plasma clearances of 81 and 59 mL/min/kg, respectively, which were similar in magnitude to the rate of hepatic blood flow. The oral bioavailability of genistein was 25% in mice that received doses of 50 mg/kg and only 4.5% in dogs given 25 mg/kg. Thus, the mean plasma concentration of the drug during the 6 hr period after po dosing was considerable higher in mice (0.16-0.35 micro g/mL) than dogs (0.052 to 0.056 micro g/mL). Dogs treated with doses of 5 mg/kg iv or 25 mg/kg po exhibited no clinical indications of drug toxicity and there were no alterations in any of the hematological or serum chemistry parameters. Additional studies will be required in order to completely characterize the pharmacokinetic behavior of genistein in preclinical animal models.